The proliferating aortic smooth muscle cell ingests and exports a variety of substances and is thought to play a significant role in plaque formation during atherogenesis. In tissue culture this cell exhibits similar active metabolic processes and is a useful model for studying membrane biogenesis. This proposal will study membrane glycoprotein biosynthesis with special emphasis on the participation of polyisoprenyl phosphates in their carbohydration. The latter are of particular significance to the smooth muscle cell since they share a common biosynthetic pathway with cholesterol. The proposal will examine the function and control of these prenols, isolate the oligosaccharide units whose assembly they mediate, and relate the latter to specific membrane constituents. Calf aorta cells will be grown in the presence of radioactive mevalonate, and pulsed with differently labeled mannose or glucosamine. Polyisoprenyl (pyro) phosphoryl saccharides will be isolated from particulate fractions according to their solubility properties in CHC13-CH30H-H20 mixtures, and purified by slica gel and DEAE chromatography Oligosaccharides will be released by mild acid hydrolysis and their fine structure will be detailed by gel filtration sizing, specific glycosidase digestion, partial hydrolyses, acetolysis, and permethylation. The labeled lipid moiety will be identified by paper and TLC. To examine the intermediary role of the prenol sugars, antibiotics such as Puromycin will be used to effect their accumulation and Bacitracin their depletion; sterols will be utilized to control their synthesis; and pulse-chase kinetics will be carried out to study their turnover. Glycopeptides will be isolated from pronase digests of membrane fractions obtained from sucrose density gradient, purified by gel and ion exchange chromatographic, and their patterns examined to define specific responses to alteration of prenol saccharide levels. Structure of particular glycopeptides will be determined as for the prenol oligosaccharide to establish identity. Finally, membranes will be solubilizied by detergents or chaotropes and specific glycoproteins will be isolated and their composition defined.